Biological transmission of rickettsia from ticks is preceded by infection of the vector, which starts with transfer of the pathogen from the blood meal in the tick midgut lumen to the midgut epithelium. Manipulation of host cell actin appears to be an important feature of this rickettsial infection pathway, and conservation of this trait among families of tick-borne rickettsia suggests that it is important for the survival of these organisms in nature. There is evidence that F-actin structures perpetuate the transmission cycle of the rickettsial family Anaplasmataceae by providing actin-based motility that facilitates contact with the tick midgut epithelium that is subsequently infected by the organisms. The objective of this application is to understand the mechanism responsible for what is likely to be the initial step in rickettsial infection of the tick host: specifically, motility in the tick blood meal. The central hypothesis of the proposed research is that the anaplasmal appendage associated protein is responsible for actin-based motility prior to pathogen invasion of tick midgut epithelial cells. We plan to test this hypothesis by pursuing the following specific aims: (1) to define the mammalian cytoplasmic constituents of anaplasmal appendages and (2) to delineate the role of appendage components in tick infection by A. marginale. The rationale for the proposed research is that, once anaplasmal actin-based motility in the blood meal is understood, it will provide new insights into transmission and possibly pathogenic mechanisms associated with these pathogens. Moreover, due to the highly conserved nature of the host proteins involved and the conservation of this trait among the Anaplasmataceae and the Rickettsiaceae, it is likely that this mechanism of vector infection will be also be conserved among the order Rickettsiales. This is expected to have significant positive effects on human health, because it will lead to exploration of new approaches such as interference with rickettsial manipulation of cytoskeletal proteins to control transmission of such pathogens at the vector infection event, which represents a key point where the rickettsial transmission cycle can be broken. PUBLIC HEALTH RELEVANCE There are critical gaps in our understanding of the molecular events that enable bacterial parasites of the order Rickettsiales to infect host cells. This project will establish how rickettsial pathogens exploit the host cell cytoskeletons to infect ticks and will enable development of new strategies to interfere with acquisition and subsequent transmission of these tick-borne pathogens.